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Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.

Tuma MC, Zill A, Le Bot N, Vernos I, Gelfand V - J. Cell Biol. (1998)

Bottom Line: Natl.Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport.We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

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Overexpression of  headless Xklp3 inhibits pigment dispersion. Cell nuclei  were injected with plasmids  pEGFP-C1 (A) or pEGFP-headless Xklp3 (B). Cells  were allowed to recover and  express exogenous protein  for 48 h and then treated with  melatonin for 1 h to induce  pigment aggregation, followed  by 1 h in MSH, to disperse  pigment. A cell expressing  EGFP (A) dispersed its pigment normally, while a cell  expressing the EGFP-headless Xklp3 fusion protein (B)  dispersed melanosomes only  partially. Bright field and fluorescence images of cells  were overlaid to show pigment distribution in cells expressing EGFP-tagged proteins. Bar, 20 μm.
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Figure 3: Overexpression of headless Xklp3 inhibits pigment dispersion. Cell nuclei were injected with plasmids pEGFP-C1 (A) or pEGFP-headless Xklp3 (B). Cells were allowed to recover and express exogenous protein for 48 h and then treated with melatonin for 1 h to induce pigment aggregation, followed by 1 h in MSH, to disperse pigment. A cell expressing EGFP (A) dispersed its pigment normally, while a cell expressing the EGFP-headless Xklp3 fusion protein (B) dispersed melanosomes only partially. Bright field and fluorescence images of cells were overlaid to show pigment distribution in cells expressing EGFP-tagged proteins. Bar, 20 μm.

Mentions: Immortalized Xenopus melanophores (a gift of Dr. Michael Lerner, University of Texas Southwestern Medical Center, Dallas, TX) were cultured at 27°C in 0.7× Leibowitz L-15 medium (GIBCO BRL; Life Technologies, Gaithersburg, MD) supplemented with 5% fetal calf serum (HyClone Labs, Logan, UT), 5 mg/ml insulin, and 100 μg/ml each of penicillin and streptomycin (see details in Daniolos et al. [1990] and Rogers et al. [1997]). For microscopic analysis, cells were plated on acid-washed poly- l-lysine–coated coverslips in serum-containing medium. 24 h before the experiment, cells were shifted to serum-free medium. Aggregation or dispersion of pigment was induced by treating melanophores with either 10 nM melatonin or 100 nM MSH, respectively, in serum-free medium for 1 h. Cells were fixed with 4% formaldehyde, and the state of pigment distribution was scored under an upright microscope (model Microphot-SA; Nikon, Inc., Melville, NY) as follows: dispersed, aggregated, and partially dispersed/aggregated (Reilein et al., 1998). Examples are shown in Fig. 3 A for dispersed and 3 B for partially dispersed; in cells considered aggregated, the pigment forms a tight mass in the cell center (not shown). A minimum of 100 cells was counted per experiment, and each experiment was repeated at least three times.


Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.

Tuma MC, Zill A, Le Bot N, Vernos I, Gelfand V - J. Cell Biol. (1998)

Overexpression of  headless Xklp3 inhibits pigment dispersion. Cell nuclei  were injected with plasmids  pEGFP-C1 (A) or pEGFP-headless Xklp3 (B). Cells  were allowed to recover and  express exogenous protein  for 48 h and then treated with  melatonin for 1 h to induce  pigment aggregation, followed  by 1 h in MSH, to disperse  pigment. A cell expressing  EGFP (A) dispersed its pigment normally, while a cell  expressing the EGFP-headless Xklp3 fusion protein (B)  dispersed melanosomes only  partially. Bright field and fluorescence images of cells  were overlaid to show pigment distribution in cells expressing EGFP-tagged proteins. Bar, 20 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132968&req=5

Figure 3: Overexpression of headless Xklp3 inhibits pigment dispersion. Cell nuclei were injected with plasmids pEGFP-C1 (A) or pEGFP-headless Xklp3 (B). Cells were allowed to recover and express exogenous protein for 48 h and then treated with melatonin for 1 h to induce pigment aggregation, followed by 1 h in MSH, to disperse pigment. A cell expressing EGFP (A) dispersed its pigment normally, while a cell expressing the EGFP-headless Xklp3 fusion protein (B) dispersed melanosomes only partially. Bright field and fluorescence images of cells were overlaid to show pigment distribution in cells expressing EGFP-tagged proteins. Bar, 20 μm.
Mentions: Immortalized Xenopus melanophores (a gift of Dr. Michael Lerner, University of Texas Southwestern Medical Center, Dallas, TX) were cultured at 27°C in 0.7× Leibowitz L-15 medium (GIBCO BRL; Life Technologies, Gaithersburg, MD) supplemented with 5% fetal calf serum (HyClone Labs, Logan, UT), 5 mg/ml insulin, and 100 μg/ml each of penicillin and streptomycin (see details in Daniolos et al. [1990] and Rogers et al. [1997]). For microscopic analysis, cells were plated on acid-washed poly- l-lysine–coated coverslips in serum-containing medium. 24 h before the experiment, cells were shifted to serum-free medium. Aggregation or dispersion of pigment was induced by treating melanophores with either 10 nM melatonin or 100 nM MSH, respectively, in serum-free medium for 1 h. Cells were fixed with 4% formaldehyde, and the state of pigment distribution was scored under an upright microscope (model Microphot-SA; Nikon, Inc., Melville, NY) as follows: dispersed, aggregated, and partially dispersed/aggregated (Reilein et al., 1998). Examples are shown in Fig. 3 A for dispersed and 3 B for partially dispersed; in cells considered aggregated, the pigment forms a tight mass in the cell center (not shown). A minimum of 100 cells was counted per experiment, and each experiment was repeated at least three times.

Bottom Line: Natl.Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport.We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

Show MeSH
Related in: MedlinePlus